Accumulated studies have shown that microvesicles (microparticles) are involved in cell-to-cell communication by transfer of bioactive molecules including proteases, mRNAs, microRNAs, membrane receptors, and organelles among cells thus play essential physiological/pathological roles including the progression of various diseases such as coagulation diseases, wound healing, atherosclerosis, coronary diseases, diabetes, hematologic diseases, infectious diseases, inflammatory diseases, neurologic diseases, and metastasis of cancer (Simak et al. 2006; Cocucci et al. 2009; Muralidharan-Chari et al. 2010). Much interest centers on microvesicles and nanovesicles (exosomes) as they are increasingly cited as potential biomarkers. Microvesicles and exosomes are differentiated both by their size ranges and their biogenesis. Microvesicles are small, plasma-membrane-derived particles that are released into the extracellular environment by the outward budding and fission of the plasma membrane. Typically microvesicles are described as being 100 nm to 15 μm, whilst exosomes are in the range 30-100 nm. Microvesicles are typically formed by blebbing of the plasma membrane, whereas exosomes are released by exocytosis from multivesicular bodies of the endosome (Simak et al. 2006; Cocucci et al. 2009; Muralidharan-Chari et al. 2010).
Invasion and metastasis of tumor cells are the major cause of cancer-related morbidity and mortality. Tumor cells produce extracellular matrix (ECM)-degradation protease to degrade ECM for their invasion and metastasis. Microvesicles are plasma-membrane-derived particles and tumor-released microvesicles are rich in ECM-degradation proteases and thus play important role in the invasion and metastasis of cancer cells (Cocucci et al. 2009). The cellular signaling pathways that regulate microvesicle generation/shedding and cancer metastasis are not fully understood. Molecularly-targeted therapy is becoming a main stream in cancer therapy. The molecularly targeted drugs inhibit tumor progression by interrupting specific cellular signaling pathways that are abnormally activated in cancer cells. Therefore, identification of the cellular signaling that control the generation or release of microvesicle may facilitate finding of target for developing of targeted drugs for cancer, and may also facilitate the identification of biomarker that can conjunction with specific molecularly-targeted therapy for patient selection, monitoring, and management.
ERK (extracellular signal-regulated kinases) has been shown to mediate microvesicle shedding in tumor invasion (Muralidharan-Chari et al. 2009). Microvesicles need to be generated before being released outside cell membrane. However, little is known about the cellular signaling pathways that regulate microvesicle generation and cancer metastasis.
CSE1L (chromosome segregation 1-like protein), also named as CAS (cellular apoptosis susceptibility protein; GenBank accession no. U33286), is highly expressed in cancer and is involved in cancer invasion and metastasis (Brinkmann et al. 1995; Tung et al. 2009; Tai et al. 2010). CSE1L has previously been shown to be a cellular tyrosine phosphorylated protein that is phosphorylated by MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase kinase) and this is related with the nuclear transport of CSE1L (Scherf et al. 1998). Microvesicles produced by tumor cells play important role in cancer metastasis (Muralidharan-Chari et al. 2010).
The ERK signaling pathway is involved in the proliferation, survival, invasion and metastasis of cancer cells (Roberts et al. 2007). Aberrant activation of the ERK pathway has been shown to be an essential feature common to many types of human tumors (Hoshino et al. 1999). The present invention discloses that CSE1L is linked to ERK signaling pathway, CSE1L is phosphorylated upon ERK activation, phosphorylated CSE1L is present in sera from cancer patients, and assay of serum phosphorylated CSE1L is superior to assay of serum CSE1L for cancer diagnosis. Therefore, serum phosphorylated CSE1L should have clinical utility in cancer diagnosis.
The Ras-ERK pathway is a potential targets for development of molecularly-targeted drug for cancer therapy (Roberts et al. 2007).
A significant limitation in molecularly-targeted therapy is that not all patients can receive targeted therapy since the patient's tumor may not express the target that the therapy is being directed to. Also, tumor-related mortalities still occur in targeted therapy due to the proliferation of the “target-negative” tumor cell population. Shedding microvesicles of specific composition may be loaded with drugs addressed with high precision to unique cellular targets (Cocucci et al. 2009). Microvesicles generation/shedding should be the common feature occurs in tumors (Simak et al. 2006; Cocucci et al. 2009; van Doormaal et al. 2009; Muralidharan-Chari et al. 2010).
U.S. Pat. No. 6,664,057 disclosed the identification of a novel amplicon on human chromosome 20q13.2 which is associated with cancer. U.S. Pat. No. 6,072,031 disclosed the cDNA and amino acid sequences for the cellular apoptosis susceptibility protein are used to detect expression and amplification of the CSE1L gene in normal and cancer cells. An antisense CSE1L gene sequence introduced into living cells inhibits CSE1L protein activity and thus prevents or inhibits apoptosis in the cells. U.S. Pat. No. 5,759,782 disclosed the cDNA and amino acid sequences for the cellular apoptosis susceptibility protein are used to detect expression and amplification of the CSE1L gene in normal and cancer cells. An antisense CSE1L gene sequence introduced into living cells inhibits CSE1L protein activity and thus prevents or inhibits apoptosis in the cells. U.S. Pat. No. 6,207,380 claims assay of polypeptides and polynucleotides in urinary tract tissue and is useful for detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the urinary tract. These sequences are derived from keratin/cytokeratin, CSE1L, or mat-8 polypeptides and polynucleotides. U.S. Pat. No. 6,207,380 also provided are antibodies that specifically bind to keratin/cytokeratin, CSE1L, or mat-8-encoded polypeptides or proteins in the urinary tract tissue, which molecules are useful for the therapeutic treatment of urinary tract diseases. Thus, U.S. Pat. No. 6,207,380 disclose using antibodies that specifically bind to keratin/cytokeratin, CSE1L, or mat-8 in the urinary tract tissue for the therapy of urinary tract diseases. U.S. Pat. No. 6,232,086 disclose the cDNA and amino acid sequences for a CSE1L protein that can be used to detect expression and amplification of the CSE1L gene in normal and cancer cells. U.S. Pat. No. 6,156,564 disclose a method of detecting human proliferating cells comprising measuring a level of a human CSE1L protein in a human cell sample and detecting the human CSE1L protein at a level at least two-fold greater than the level of a human CSE1L protein in normal non-proliferating human cells. U.S. Pat. No. 6,440,737 disclose antisense compounds, compositions and methods for modulating the expression of CSE1L gene. The disclosure of US20080081339 describes measuring dozens of autoantibodies including CSE1L autoantibody (not antigens or tumor associated marker) present in a body fluid as biomarkers for prostate cancer diagnosis. The disclosure of US20050260639 describes determining the expression level of pancreatic cancer-associated gene including CSE1L in circulation cancer cells isolated from bodily fluid or bodily tissue for detecting and diagnosing pancreatic cancer. Based on the result of a cDNA gene chip, the disclosure of WO2009/052573 describes isolating hundreds of RNA transcripts including CSE1L transcript (i.e. CSE1L mRNA) from circulation tumor cells in blood sample or tissue to assay the onset of an adenoma state of gastrointestinal cancer. The disclosure of US20100120074 describes the measurement of CSE1L or CSE1L polypeptide levels in the body fluids for metastatic cancer diagnosis. US20110053157 describes the measurement of nucleic acids including DNA, RNA, and microRNA in microvesicles (exosomes) for diagnosis, prognosis and treatment of medical diseases and conditions. US20110053157 describes methods of aiding diagnosis, prognosis, monitoring and evaluation of a disease or other medical condition in a subject by detecting a DNA or RNA biomarker in microvesicles isolated from a biological sample from the subject; method of delivering a nucleic acid or protein to a target all by administering microvesicles that contain said nucleic acid or protein; methods for performing a body fluid transfusion by introducing a microvesicle-free or microvesicle enriched fluid fraction into a patient. WO2009021322 and US20100255514 describe method for diagnosis and prognosis of cancer and for monitoring the progression of cancer and/or the therapeutic efficacy of an anti-cancer treatment in a sample of a subject by detecting selected oncogenic proteins consisting of EGFRvIII, EGFR, HER-2, HER-3, HER-4, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, PI3K, Akt, BRCA1, BRCA2, PTEN, and receptors of cells associated with cancer (cancer-related receptors) such as VEGFR-2, VEGFR-1, Tie-2, TEM-1 and CD276 in microvesicles. The prior arts above detect the expression of CSE1L gene in cell level or CSE1L protein level in the body fluids. Therefore, none of the references describes or claim CSE1L or phosphorylated CSE1L binding agents, such as anti-CSE1L antibody or anti-phosphorylated CSE1L antibody and derivatives as well as pharmaceutical compositions and kits comprised the antibodies or derivative for detecting CSE1L or phosphorylated CSE1L in microvesicles or fluids from biological samples. Also, no prior arts disclosed, described, or claimed methods for the treatment and prevention of diseases comprised administering to a subject the CSE1L-binding agent or the phosphorylated CSE1L-binding agent. Also, no references disclosed, described, or claimed methods and kit comprising CSE1L-binding agent or phosphorylated CSE1L-binding agent for microvesicle isolation, analysis, or targeting for disease diagnosis or treatment.